Qian, C. (Cheng)
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- Alpha(v)beta(3) integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment(Nature Publishing Group, 2001) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Qian, C. (Cheng); Bilbao, R. (Roberto); Melero, I. (Ignacio); Narvaiza, I. (Íñigo); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Bustos, M. (Matilde)We previously reported that systemic injection of recombinant adenovirus resulted in a rim of gene transduction around experimental liver tumor nodules. This zone of higher infection is dependent on the alpha(v)beta(3) integrin, acting as an adenovirus internalization receptor, which is overexpressed in tissues surrounding liver metastases. When a recombinant adenovirus encoding interleukin-12 (AdCMVIL-12) is given into a subcutaneous tumor nodule in mice also bearing concomitant liver tumors, a fraction of AdCMVIL-12 reaches the systemic circulation and infects liver tissue, especially at the malignant/healthy tissue interface. As a result of the expression at this location of the interleukin-12 transgenes, VCAM-1 is induced on vessel cells and mediates the recruitment of adoptively transferred anti-tumor cytolytic T-lymphocytes. These studies provide mechanistic explanations for the potent therapeutic synergy observed between interleukin-12 gene transfer and adoptive T-cell therapy.
- The stilbene disulfonic acid DIDS stimulates the production of TNF-alpha in human lymphocytes(Elsevier, 1992) Civeira, M.P. (María Pilar); Arrazola, A. (Arantxa); Larrea, E. (Esther); Medina, J.F. (Juan Francisco); Diez-Martinez, J. (Javier); Qian, C. (Cheng); Prieto, J. (Jesús); Garciandia, A. (Ana)Exposure of human peripheral blood mononuclear cells (PBMC) to the stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (60 microM and above) significantly increased the release of tumor necrosis factor-alpha (TNF-alpha), as determined by TNF-alpha activity in the incubation media. When the TNF-alpha message was analyzed in PBMC by a reverse transcription/polymerase chain reaction (RT/PCR)-based procedure, it was found that incubation with DIDS (60 microM) was followed by a time-dependent accumulation of TNF-alpha mRNA. Measurements of intracellular pH showed that the presence of increasing concentrations of DIDS resulted in a progressive intracellular alkalinization of PBMC. It is suggested that the known DIDS effect of inhibiting transmembrane anion exchange, i.e., chloride/bicarbonate exchange, might play a role in the stimulation of TNF-alpha production by PBMC exposed to DIDS.
- Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens(Wiley-Blackwell, 2004) Mazzolini, G. (Guillermo); Alfaro, C. (Carlos); Duarte, M. (Marina); Qian, C. (Cheng); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Feijoo, E. (Esperanza); Chen, L. (Lieping); Arina, A. (Ainhoa)Intralesional administration of cultured dendritic cells (DCs) engineered to produce IL-12 by in vitro infection with recombinant adenovirus frequently displays eradicating efficacy against established subcutaneous tumors derived from the CT26 murine colon carcinoma cell line. The elicited response is mainly mediated by cytolytic T lymphocytes. In order to search for strategies that would enhance the efficacy of the therapeutic procedure against less immunogenic tumors, we moved onto malignancies derived from the inoculation of MC38 colon cancer cells that are less prone to undergo complete regression upon a single intratumoral injection of IL-12-secreting DCs. In this model, we found that repeated injections of such DCs, as opposed to a single injection, achieved better efficacy against both the injected and a distantly implanted tumor; that the use of semiallogeneic DCs that are mismatched in one MHC haplotype with the tumor host showed slightly better efficacy; and that the combination of this treatment with systemic injections of immunostimulatory anti-CD137 (4-1BB) monoclonal antibody achieved potent combined effects that correlated with the antitumor immune response measured in IFN-gamma ELISPOT assays. The elicited systemic immune response eradicates concomitant untreated lesions in most cases. Curative efficacy was also found against some tumors established for 2 weeks when these strategies were used in combination. These are preclinical pieces of evidence to be considered in order to enhance the therapeutic benefit of a strategy that is currently being tested in clinical trials. Supplementary Material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.
- Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL)(Nature Publishing Group, 1998) Qian, C. (Cheng); Bilbao, R. (Roberto); Prieto, J. (Jesús); Drozdzik, M. (Marek); Lasarte, J.J. (Juan José)Fas ligand is a type II transmembrane protein which can induce apoptosis in Fas-expressing cells. Recent reports indicate that expression of FasL in transplanted cells may cause graft rejection and, on the other hand, tumor cells may lose their tumorigenicity when they are engineered to express FasL. These effects could be related to recruitment of neutrophils by FasL with activation of their cytotoxic machinery. In this study we investigated the antitumor effect of allogenic fibroblasts engineered to express FasL. Fibroblasts engineered to express FasL (PA317/FasL) did not exert toxic effects on transformed liver cell line (BNL) or colon cancer cell line (CT26) in vitro, but they could abrogate their tumorigenicity in vivo. Histological examination of the site of implantation of BNL cells mixed with PA317/FasL revealed massive infiltration of polymorphonuclear neutrophils and mononuclear cells. A specific immune protective effect was observed in animals primed with a mixture of BNL or CT26 and PA317/FasL cells. Rechallenge with tumor cells 14 or 100 days after priming resulted in protection of 100 or 50% of animals, respectively. This protective effect was due to CD8+ cells since depletion of CD8+ led to tumor formation. In addition, treatment of pre-established BNL tumors with a subcutaneous injection of BNL and PA317/FasL cell mixture at a distant site caused significant inhibition of tumor growth. These data demonstrate that allogenic cells engineered with FasL are able to abolish tumor growth and induce specific protective immunity when they are mixed with neoplastic cells.
- Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice(Wiley-Blackwell, 2006) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Rooijen, N. (Nico) van; Qian, C. (Cheng); Kochanek, S. (Stefan); Barajas, M. (Miguel); Shankar, V. (Vijay); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Mauleon, I. (Itsaso)Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.
- Hepatic and extrahepatic HCV RNA strands in chronic hepatitis C: different patterns of response to interferon treatment(Wiley-Blackwell, 1993) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Qian, C. (Cheng); Gil, B. (Beatriz); Prieto, J. (Jesús)We investigated the presence of positive (genomic) and negative (replicative intermediate) hepatitis C virus RNA strands in liver, peripheral mononuclear cells and serum from patients with chronic hepatitis C using a selective and semiquantitative polymerase chain reaction procedure. Negative and positive hepatitis C virus RNA strands were present in liver, serum and lymphoid cells in all untreated patients and in all those who did not respond to interferon therapy. In the latter group of patients, the titers of RNA strands in the liver and peripheral mononuclear cells at the end of the treatment were similar to those encountered in untreated patients, but the serum titers were about 100 times lower than pretreatment values. In patients who responded to interferon with normalization of serum aminotransferase levels (n = 10), the rate of detection and the titer of the two viral strands in liver, serum and mononuclear cells were markedly decreased at the end of the therapy. In the six responders who did not relapse after interferon withdrawal, both hepatitis C virus RNA strands were absent from the liver, serum and lymphoid cells. By contrast, the positive RNA strand was present in liver cells, mononuclear cells or both at the end of therapy in all patients who experienced posttherapy relapse. In conclusion, our results indicate that interferon can clear hepatitis C virus from hepatic and extrahepatic sites only in responder patients. Disappearance of genomic hepatitis C virus RNA from the liver and from mononuclear cells may predict complete response without posttherapy relapse.
- Taurocholate-stimulated leukotriene C4 biosynthesis and leukotriene C4-stimulated choleresis in isolated rat liver(WB Saunders, 1995) Medina, J.F. (Juan Francisco); Vesperinas, I. (Idoia); Rodriguez-Ortigosa, C.M. (Carlos M.); Qian, C. (Cheng); Prieto, J. (Jesús); Quiroga, J. (Jorge)BACKGROUND/AIMS: Cysteinyl-containing leukotrienes seem to exert a cholestatic effect. However, leukotriene inhibitors were found to reduce bile salt efflux in isolated rat hepatocytes, suggesting a role for leukotrienes in bile flow formation. METHODS: In the isolated rat liver, the effects of two different concentrations of leukotriene C4 on bile flow and bile salt excretion are analyzed, as well as the possible effect of taurocholate on the hepatic production of cysteinyl-containing leukotrienes. RESULTS: Leukotriene C4 (0.25 fmol) increased bile salt excretion (+22.2%; P < 0.05), whereas a much higher dose (0.25 x 10(6) fmol) showed the known cholestatic effect, reducing bile salt excretion (-25.9%; P < 0.01). These dose-dependent biphasic effects were specific because they could be prevented by the simultaneous administration of cysteinyl-containing leukotriene antagonists. On the other hand, taurocholate administration induced a dose-dependent increase in biliary excretion of cysteinyl-containing leukotrienes. Furthermore, taurocholate increased messenger RNA levels of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Taurocholate increase of hepatocyte intracellular calcium was not significant, suggesting that taurocholate effects are not mediated by stimulation of calcium metabolism. CONCLUSIONS: These results constitute evidence for the existence of a positive feedback mechanism by which bile salts stimulate the synthesis of leukotrienes that, in turn, stimulate bile salt excretion.
- Liver Damage using Suicide Genes A Model for Oval Cell Activation(Elsevier, 2000) Qian, C. (Cheng); Beraza, N. (Naiara); Sangro, B. (Bruno); Alzuguren, P. (Pilar); Gil, A.G. (Ana Gloria); Garcia-Pardo, A. (Ángeles); Prieto, J. (Jesús); Bustos, M. (Matilde); Ruiz, J. (Juan)Liver regeneration from the facultative hepatic stem cells, the oval cells, takes place in situations in which liver regeneration from pre-existing hepatocytes is prevented. Different models have been used to stimulate oval cell response. Many of them involve the use of carcinogenic agents with or without partial hepatectomy. In this study we show that adenovirus-mediated gene transfer of the suicide gene thymidine kinase followed by ganciclovir administration caused hepatotoxicity of variable intensity. Rats with moderate elevation in serum transaminases recovered normal liver architecture few weeks after adenovirus injection. In contrast, rats with severe liver damage exhibited a marked and persisting activation of oval cells accompanied by ductular hyperplasia. In some rats, such lesion eventually evolved to cholangiofibrosis and in one rat to cholangiocarcinoma. Deposition of fibronectin and increased number of hepatic stellate cells were found in association with oval cells and cholangiofibrotic lesions. Hepatocyte growth factor was hyperexpressed in the livers with intense oval cell response or ductular proliferation, suggesting a participation of this factor in those lesions. In summary, our data demonstrate activation of oval cell response after gene transfer of thymidine kinase followed by ganciclovir administration. These findings indicate that high doses of this therapy causes liver damage together with an impairment in hepatocellular regeneration.
- Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy(American Association of Immunologist, 2000) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Qian, C. (Cheng); Zaratiegui, M. (Mikel); Barajas, M. (Miguel); Melero, I. (Ignacio); Narvaiza, I. (Íñigo); Prieto, J. (Jesús)We have constructed a recombinant defective adenovirus that expresses functional murine IFN-gamma-inducible protein-10 (IP-10) chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar s. c. CT26 tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor regression in nearly 70% of cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication not only against injected, but also against distant noninjected tumor nodules. Colocalization of both adenoviruses at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor-specific CTL response in a synergistic fashion, while both CD4 and CD8 T cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD4+ and CD8+ T cells but was largely unaffected by single depletion of each T cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit one to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12-mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.
- Gene therapy of cancer based on interleukin 12(Bentham Science Publishers, 2005) Qian, C. (Cheng); Sangro, B. (Bruno); Melero, I. (Ignacio); Prieto, J. (Jesús)Tumor formation and growth depends mainly on the inability of the organism to elicit a potent immune response, and on the formation of new blood vessels that enable tumor nutrition. Interleukin-12 (IL-12) therapy can target both processes. And IL-12-based gene therapy may restrict IL-12 production to the relevant site in order to obtain enhanced antitumor activity and reduced toxicity. In the clinical setting, IL-12 gene transfer can be used either to improve the pharmacokinetic/pharmacodynamic profile of the cytokine, to transduce dendritic cells or to enhance the efficiency of antitumor vaccination. It can also synergize with other procedures involving the simultaneous transfer of other transgenes or non-gene based strategies. The strong anti-tumoral power shown in many different animal models has not been found in early clinical trials in which cancer patients were treated by peritumoral injections of autologous fibroblasts producing IL-12, intratumoral injections of an adenoviral vector encoding human IL-12 genes, or intratumoral injection of autologous dendritic cells transduced ex vivo with this same adenoviral vector. However, these trials have set the proof-of-concept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells and that in some cases it is followed by tumor regression. From the many questions that arise after these disappointing results the most relevant concerns the duration and intensity of transgene expression and the capability to monitor this topics in vivo. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing.